The etiology of essential hypertension is complex and largely unknown, but one of its hallmark features is elevated Ca2+-dependent vascular tone. The vascular smooth muscle cells (VSMCs) of the arterial wall express L-type calcium (CaL) channels in their plasma membranes. Depolarization of VSMCs opens the CaL channels to elicit vasoconstriction. This process is an important regulator of the peripheral vascular resistance which, in turn, determines systemic blood pressure. The vascular CaL channel appears to be minimally composed of a pore-forming α 1C subunit and accessory β and α2δ subunits. Studies in neuronal tissues and heterologous expression systems suggest that the β subunits are required for the trafficking of α 1C subunits from the endoplasmic reticulum to the plasma membrane to form functional CaL channels. There are four known CaL channel β subunits (β1-4) that are expressed in a tissue-dependent manner. In large arteries, the CaL channel α1C subunit appears to associate with β2 or β3 subunits that are required for its normal surface expression, but the exact identity and role of the β subunit(s) expressed in small resistance arteries is not well defined. In the present study, our goal was to identify the types of β subunit(s) in mouse mesenteric VSMCs and define their contribution to CaL channel expression in the small mesenteric arteries. To identify the β subunit(s), multi-cell nested PCR was performed using mRNA isolated from collections of mesenteric VSMCs and specific primer sets. Only signals for β2 and β3 transcripts were amplified in the samples from mesenteric VSMCs. Subsequent immuno blots using specific antibodies only confirmed expression of the β3 subunit at the protein level. Next, we found that only the CaL channel β3 subunit was selectively coupregulated with the pore-forming α 1C subunit in mesenteric arteries of angiotensin II (Ang II)-infused hypertensive mice. Thus, we propose that the β3 subunits positively regulate vascular CaL channel expression and levels of blood pressure in vivo, and that the pressure-induced upregulation of vascular CaL channels relies on the presence of this critical subunit. In order to directly test the role of the β3 subunit in the upregulation of CaL channels during hypertension, we established a colony of β3KO mice. Western blots revealed that the mesenteric arteries from β3KO mice expressed 30±8 % less α1C subunit protein compared to WT mice (n=5). Initial single channel patch-clamp recordings suggested that the VSMCs from β3KO mice expressed comparable functional CaL channels to WT mice at the plasma membrane. Surprisingly, the resting systolic blood pressures were similar between WT and β3KO mice. Next, we infused WT and β3KO mice with saline or Ang II (2 ng/g/min) for 2 weeks to assess if the β3 subunit is required for the upregulation of vascular Ca L channels during the development of hypertension. Both α 1C and β3 subunit proteins were upregulated in mesenteric arteries of WT mice infused with Ang II compared to saline-infused WT mice. In contrast, the upregulation of α1C in response to Ang II was significantly blunted in mesenteric arteries of β3KO mice compared to the WT animals. Isolated, cannulated mesenteric arteries from Ang II-infused β3KO mice also showed a reduced contractile sensitivity to the CaL channel agonist FPL 64176. Finally, at the end of two weeks of Ang II infusion, the β3KO mice showed significantly lower systolic blood pressures compared to Ang II-infused WT mice (152±5 mm Hg in β3KO vs. 180±5 mm Hg in WT). As a final aim, we established a new method to monitor the turnover rate of Ca L channels in rat mesenteric arteries in situ. A portion of mesenteric arterial bed contained in an intestinal loop of a rat was exteriorized in a perfusion chamber and exposed for different durations to either control physiological salt solution (PSS) or PSS containing puromycin (20 μg/ml) to inhibit de novo protein synthesis. Mesenteric arterial proteins were collected to assess vascular CaL channel expression. Western blot analysis revealed that the α1C subunit protein in rat mesenteric arteries was stable in control PSS for 6 hours using this preparation, but was rapidly degraded in the presence of puromycin. In the arteries exposed to puromycin, α 1C expression was reduced by nearly 75% in 6 hours compared to untreated arteries. A similar result was observed in initial studies using mouse mesenteric intestinal loops. Our findings suggest that vascular CaL turnover is highly dynamic. Thus, we have a model in which to explore the stimuli (e.g. blood pressure), proteins (e.g. β3 subunit) or other mechanisms that may regulate vascular CaL channel expression in vivo. In summary, our findings suggest that: i) β3 may be the sole β subunit expressed in mouse small mesenteric arteries, ii) the β3 subunit is involved in the upregulation of vascular CaL channels during hypertension, and iii) CaL channel expression is highly dynamic and our newly developed model will enable researchers to identify the factors that regulate channel expression in vivo. Funding AHA 09PRE2250224 (SVK) and NIH R01 HL064806-10 (NJR).
|Advisor:||Rusch, Nancy J.|
|Commitee:||Jaggar, Jonathan H., Macmillan, Lee Ann, Marsh, James D., Mayeux, Philip R., Rhee, Sung W.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 72/12, Dissertation Abstracts International|
|Keywords:||Hypertension, Mesenteric arteries, Vascular calcium channels|
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